RNA:protein interactions regulate many aspects of the growth and metabolism of mammalian cells and, in particular, regulate critical stages in the replication of many pathogenic human viruses. The investigation of such binding events, however, has historically been restricted largely to in vitro, that is, cell free, assays that may not always be able to recapitulate in vivo interactions accurately. A three hybrid system for measuring RNA:protein interactions in vivo has been described (SenGupta et al, Proc. Natl. Acad. Sci. USA 93:8496 (1996)). The system used, however, is a yeast system. As a result, specific protein post-translational modifications that may be critical for RNA binding in mammalian cells, and that may not be possible in the yeast system, may not occur as normal. Further, any mammalian cofactors required for RNA binding, may not be expressed in the yeast system.
The present invention provides a mammalian-cell based RNA binding assay useful for the effective demonstration and characterization of numerous RNA:protein interactions. The effectiveness of the assay is exemplified by the use of the system to characterize the interaction of picornavirus 3C proteases with their cognate RNA target sequences in vivo, and to characterize the interaction of Mason-Pfizer Monkey Virus (MPMV) constitutive transport element (CTE) mutants with human Tap protein.
Picornaviruses contain a positive-sense RNA genome of .about.7500 nt that is translated into a single polyprotein and are therefore dependent on proteolytic processing by virally encoded proteinases for the generation of mature viral gene products (Dougherty et al, Microbial. Rev. 57:781-822 (1993)). The majority of cleavage events are mediated by the 3C proteinase or one of its precursors, 3CD, which consists of the viral RNA polymerase (3D) fused to the 3C proteinase (Hanecak et al, Proc. Natl. Acad. Sci. USA 79:3973-3977 (1982); Jore et al, J. Gen. Virol. 69:1627-1636 (1988); Ypma-Wong et al, Virology 166:265-270 (1988)). 3C cleaves primarily at Gln-Gly amino acid pairs, but additional primary sequence determinants (Nicklin et al, Biotechnology 4:36-42 (1986); Pallai et al, J. Biol. Chem. 264:9738-9741 (1989); Blair et al, J. Virol. 65:6111-6123 (1991)) and structural determinants (Ypma-Wong et al, J. Biol. Chem. 63:17846-17856 (1988b)) are required for efficient substrate recognition and cleavage by 3C activity.
In addition to its vital role as a proteinase, 3C is thought to have a key role in viral RNA replication. Picornaviruses are characterized by a large (.about.750 nt) 5' noncoding region (5' NCR), which contains sequences that direct the efficient translation of picornaviral RNAs (the internal ribosome entry site or IRES) (Pelletier et al, Nature 334:320-325 (1988); Jang et al, J. Virol. 62:2636-2643 (1988); for review, see Ehrenfeld et al, Curr. Topics Microbiol. Immunol 203:65-83 (1995)) as well as sequence elements that are required for viral RNA replication (Andino et al, Cell 63:369-380 (1990); Borman et al, EMBO J. 13:3149-3157 (1994); Shiroki et al, J. Virol. 69:6825-6832 (1995)). 3C-containing ribonucleoprotein (RNP) complexes that are required for viral replication have been proposed to form on the 5' NCRs of at least three picornaviruses, i.e. poliovirus, rhinovirus and hepatitis A virus (Andino et al, Cell 63:369-380 (1990); Leong et al, Virology 89:484-493 (1993); Kusov et al, RNA 3:291-302 (1997)). The biological relevance of this RNP complex is most evident for poliovirus and is supported by the following genetic and biochemical data: 1) 3CD can be detected in RNA replication complexes in poliovirus infected cells (Lundquist et al, Virology 89:484-493 (1978); Dasgupta et al, Proc. Natl. Acad. Sci. USA 76:2679-2683 (1979)); 2) poliovirus 3CD forms stable RNP complexes with the 5' terminal sequences of its cognate genomic RNA in vitro (Andino et al, Cell 63:369-380 (1990); 3) site-specific mutations in the 5' terminal sequences of poliovirus RNA which affect 3CD RNP complex formation in vitro result in RNA replication defects in vivo (Andino et al, Cell 63:369-380 (1990); Andino et al, EMBO J. 12:3587-3598 (1993); 4) amino acid substitution mutations in poliovirus 3C sequences have been described that affect viral RNA replication rather than 3C-mediated protein processing in vivo (Dewalt et al, In: Semler & Ehrenfeld, eds., Molecular Aspects of Picornavirus Infection and Detection (1989); Blair et al, Virology 218:1-13 (1996)); and, 5) a mutation in 5' terminal sequences of poliovirus genomic RNA that affects viral RNA replication can be suppressed by pseudoreversion mutations in 3C proteinase sequences (Andino et al, J. Virol. 64:607-612 (1990)). Therefore, poliovirus 3C, and most likely other picornavirus 3C proteinases, play a direct role in viral RNA replication that is distinct from their role in protein processing.
The 3CD proteinase derived from poliovirus type 1 (PV1) has been shown to specifically interact in vitro with a cloverleaf-like RNA secondary structural element located in the 5' terminal .about.100 nt of poliovirus genomic RNA (FIG. 1A) (Andino et al, Cell 63:369-380 (1990)). Efficient PV1 3CD/RNA complex formation in vitro depends on the presence of a cellular protein of approximately 36 kDa in size (p36) (Andino et al, Cell 63:369-380 (1990), Andino et al, EMBO J. 12:3587-3598 (1993)) or additional viral polypeptides (i.e., 3AB) (Harris et al., J. Biol. Chem. 269:27004-27014 (1994); Xiang et al, J. Virol. 69:3658-3667 (1995)). Genetic and biochemical studies indicate that the p36 cellular protein interacts with stem-loop b, while. 3CD interacts with stem-loop d within the predicted cloverleaf-like RNA secondary structural element (Andino et al, EMBO J. 12:3587-3598 (1993)). Similarly, human rhinovirus 14 (HRV14) 3C interacts with stem-loop d within the corresponding RNA cloverleaf secondary structure element present at the 5' terminus of rhinovirus genomic RNA (Walker et al, J. Biol. Chem. 270:14510-14516 (1995)). However, in contrast to PV1 3CD, HRV14 3C interacts efficiently with its RNA target in vitro in the absence of additional viral or cellular polypeptides (Leong et al, Virology 89:484-493 (1993); Walker et al, J. Biol. Chem. 270:14510-14516 (1995)). Furthermore, PV1 3CD exhibits a low affinity for RNA sequences derived from the 5' terminus of HRV 14 genomic RNA (Xiang et al, J. Virol. 69:3658-3667 (1995)), while HRV14 3C exhibits similar affinities for both PV1 and HRV14 5' terminal sequences in vitro (Walker et al, J. Biol. Chem. 270:14510-14516 (1995)). Consistent with these latter observations, poliovirus RNA replication in vivo is inhibited by substitution of HRV14 5' NCR sequences containing stem-loop d (Rohll et al, J. Virol. 68:4284-4391 (1994)). In contrast, chimeric PV1/HRV14 genomic RNAs containing the PV1 NCR and encoding an HRV 14 polyprotein replicate efficiently (Todd et al, Virology 229:90-97 (1997)). Therefore, although the PV1 and HRV14 3C proteinases exhibit similar RNA binding properties, differences exist in RNA target specificity and/or the requirements for 3C RNP complex formation. Using the in vivo RNA:protein binding assay of this invention it has been possible to further characterize the interaction of 3C with picomavirus RNA sequences in mammalian cells and to demonstrate that the poliovirus and rhinovirus 3C proteinases are in fact sufficient to bind to 5' terminal sequences derived from their respective genomic RNAs in vivo.
As indicated above, the effectiveness of the present assay has been exemplified by the use of the system to characterize the interaction of MPMV CTE mutants with human Tap protein. CTE encoded by simian type D retroviruses directs unspliced viral RNAs into a sequence-specific nuclear RNA export pathway that is at least in part similar to the pathway used by cellular mRNAs. The human Tap protein has been shown to bind the CTE, and to enhance CTE function in semi-permissive Xenopus oocytes, and has therefore been proposed as a CTE cofactor. Using the in vivo assay of the invention, it has been possible to demonstrate that the ability of the CE mutants to bind Tap closely correlates with their function in both human cells and in human Tap expressing avian cells. Using the present assays, it has also been possible to define a novel sequence-specific RNA binding motif of the Tap protein that is sufficient for CTE binding but not adequate to rescue CTE function; the isolated Tap RNA binding domain in fact acts as a dominant negative inhibitor of full-length Tap.